Dendritic cells of the immune system act as sensors of the environment through their integration of many inputs, including pathogen-derived ligands, immune cytokines and chemokines, and endogenous signals from tissues. A key question and our long-term goal is to understand how mammalian dendritic cells translate these diverse signals into specific outputs that give rise to distinct immune responses. In this proposal, we describe the application of a recently constructed genome-wide RNAi lentiviral library to rapidly silence and determine the functions of several thousand genes in dendritic cell activation after exposure to pathogenic ligands. Our preliminary studies demonstrate that this lentiviral RNAi library can be used to silence genes in both a human monocytic/dendritic cell line and mouse bone marrow dendritic cells (BMDC). In addition, we describe a pilot genetic screen that identified known and novel regulators of phagocytosis and cytokine production. We propose to: 1) develop specific and robust assays for cytokine and costimulator expression in bone marrow dendritic cells under library screening conditions; 2) screen bone marrow dendritic cells for genes that are required for their maturation using a sublibrary of RNAi viruses that target approximately 2000 genes that were selected from several sources, including extensive gene expression data from our lab, known pathogen-sensing pathway genes and their homologs, 'hits' from a pilot RNAi screen in THP-1 cells and an unbiased collection of kinases and phosphatases; 3) evaluate the role of the identified genes in several key steps of DC maturation. RNAi-based screening represents a new strategy to search the human and mouse genomes for the key genes that can fill in gaps in our understanding and open up new and unexpected areas - in analogy with genetic screens in lower organisms. The described approach will thus allow us to rapidly generate a more complete understanding of pathogen-sensing mechanisms, which in turn will enable us to design more rational immunotherapeutics and vaccines that can guide the immune system at the initial stages of an immune response to prevent or treat infections, autoimmune diseases and other immune-related disorders. [unreadable] [unreadable] [unreadable]